Method for stromal corneal repair and refractive alteration

ABSTRACT

A method and means of providing stromal repair and improved refractive correction by creating corneal stromal collagen tissue with fibril diameter and spacing that duplicates the optical transmission and diffusion characteristics of natural corneal collagen. The repair method includes implanting the collagen scaffold during laser corneal ablation or other interlamellar surgery to improve visual acuity or to preclude the possibility of ectasia

CROSS REFERENCE TO RELATED APPLICATIONS

This is a DIVISIONAL of application Ser. No. 10/414,796 filed on Sep.28, 2005

This application claims patent priority of provisional PatentApplication Ser. No. 60/373,725, filed Apr. 16^(th), 2002.

In addition, Disclosure Documents Nos. 502428 and 503243 filed Dec.7^(th), 2001 and Dec. 29^(th), 2001 respectively.

FEDERALLY SPONSORED RESEARCH

Not applicable

SEQUENCE LISTING OR PROGRAM

Not Applicable

BACKGROUND OF THE INVENTION

1. Field

This invention relates in general to corneal reconstruction and inparticular to a method and means of regenerating a corneal lamellamembrane in an effort to restore vision in patients suffering fromfailed Laser Corneal Ablation Procedure (LCAP) such as those describedas LASIK or LASEK, radial keratomy, keratoconus, corneal abrasions, andtrauma. Further, this invention holds promise as a method to devise aintegral refractive correcting contact-like lens which can be implantedon top of or into the corneal stroma.

2. Prior Art

Corneal damage is a leading cause of impaired vision and blindness.Scarring due to chemical burns, missile damage, genetic disorders,radial keratomy, or failed LCAP are leading causes of corneal eyedamage. In particular, failed LCAP is the most common source of visionloss due to corneal damage. Refractive complications can include toomuch or too little correction, or an imbalance in correction between theeyes. In some cases, patients who experience improper LCAP may be leftnear or farsighted or with astigmatism, necessitating spectacles orcontact lens wear, or in severe cases, may be faced with blindness.Corneal inflammation is another side effect, which can cause a swellingknown as diffuse interface keratitis, leading to corneal hazing, andultimately, blurred vision. LCAP performed on certain patients withlarge pupil diameters, thin corneas, or keratoconus, leading to nightglare, starbursting, haloes, reduced vision under dim lighting,blurring, or reduced overall visual acuity. At present, only cornealtransplants or penetrating keratoplasty, are considered a viabletreatment.

Given the enormous media attention given to LCAP, most individualsreadily embrace LCAP as a cure-all solution to disposing of theirglasses and contact lenses. However, all ophthalmologists readily admit,in their FDA-mandated informed consent that not everyone sees wellenough after a LCAP procedure to truly eliminate their use of glassesand contact lenses. In fact, studies have shown that over 2 percent ofLCAP patients experience degradation in visual acuity that wasuncorrectable through refractive means. Of these patients, debilitatingeffects due to irregular astigmatism and double vision (due to cornealwarping) were common. This is particularly troublesome since, unlikecataract surgery, which restores vision in defective eyes, LCAP is anelective process practiced on healthy eyes. While LCAP is certainly apreferable procedure over radial keratotomy, the success of theprocedure and the coupling of medicine and marketing has caused in manypatients, who should not have undergone the process to be largelyforgotten. Further, intraoperative complications include decenteredablations and flap complications, such as a partial or lost flap.

Postoperative effects due to failed LCAP can include pain as a result ofdisturbance of the epithelial layer, displacement of the corneal flap,inflammation, or infection. Diffuse interface or lamellar keratitis,also known as ‘DLK’ or Sands of Sahara, is the most serious reaction andcan produce corneal hazing, blurred vision, farsightedness, astigmatism,and permanent corneal irregularities. Another equally seriouscomplication is keratoectasia induced by LCAP. Ectasia is the distensionof the cornea due to an internal pressure gradient causing the cornea tosteepen and distort. The most common side effects of LCAP are dryness ofthe eyes, night glare, starbursting, haloes, induced sphericalaberration, induced coma, and reduced visual acuity. Previous attemptsto correct the corneal structure to alleviate the aforementionedconditions have been hampered by the fact that only a fixed quantity oftissue is available for ablative modification. By its' very nature,laser ablation or LCAP removes healthy tissue, thus undermining thestructural integrity of the cornea. Replacement tissue is not availabledue to the fact that no other part of the body has the specializedcollagen fibril structure inherent in the cornea.

The most widely practiced means of corneal repair has been the cornealtransplant. However, problems of tissue rejection, of immunosuppressivemedication, gross refractive errors, and limited supplies of suitabledonor tissue hamper transplants. While numerous experiments have beenconducted in an effort to create laboratory-grown corneal tissue invitro, the drawback of most of these methods is that they attempt togenerate only one type of corneal cell structure, such as the epithelialor endothelial layers. Stromal creation in the laboratory has in thepast been met with limited success since no means have been found thatsuccessfully form the delicate collagen fibrils with micron sizeddiameters and fibril spacing necessary for corneal transparency anddiffusive permeability.

Many prior art techniques rely on implanting a polymer of material(other than collagen or collagen that is devoid of fibrils), thuslacking in permeability as well as transparency inherent in nativetissue. For example, U.S. Pat. No. 4,505,855 to Bruns and Gross issuedMar. 19, 1985, describes the fabrication of a non-fibrilized collagenbutton produced by ultracentrifugation for transplantation. This conceptsuffers from the fact that the lack of a controlled fibril diameter andfibril organizational structure significantly hinders the osmoticpumping of proteins and aqueous media through the fabricated collagenregion. The same holds true with gaseous diffusion. As a result,transparency will be impaired. Further, since the collagen button isdesigned to replace only the damaged corneal stroma, leaving out othervital tissues (the stroma is responsible for 90% of corneal thickness,composed of collagen fibrils and is the principal supportive structureof the cornea. Covering the stroma is the epithelium, a cellularmembrane about 5 layers thick, below which is the Bowman's Layer, a thinlayer separating epithelium and stroma. On the anterior portion of thestroma is the endothelium layer, responsible for dehydrating the corneavia a sodium-potassium pump mechanism and to maintain corneal opticalclarity. Last is the Descemet's membrane, which is the endotheliumbasement membrane. All these layers are all conspicuously absent inBruns et al. Also, since the source of collagen is not exclusively fromthe patient or a sterile genetically engineered source, the possibilityof a gross immunologic reaction is significant.

Published U.S. Patent Application No. 88307687 to Werblin and Patel,describes a lens produced from a hydrogel material that is insertedunder a corneal cap. As indicated in U.S. Pat. No. 4,505,855 to Bruns etal, dated Mar. 19, 1985, any material that is not identical to nativetissue can and will affect optical clarity and diffusive capacityrequired for a healthy corneal structure.

Again, any means of producing a polymer implant which reduces thediffusion rate of oxygen, lipids, or aqueous media, reduces theeffectiveness of the implant. Subtle changes in the intraocular pumpingmechanism can cause significant loss in visual acuity. As before,nonnatural polymers can be rejected by the immune system.

Similar implants are revealed in prior art such as that described inEuropean Patent No. 443,094/EP B1 to Kelman & DeVore. They utilizepolymerized collagen material in conjunction with a periphery offibrilized collagen. While providing improvements over simple collagenor other polymer implants, this suffers from the fact that thepolymerized collagenous core does not contain fibrils at all as nativetissue. Moreover, the fibrils on the periphery are not of the samediameter as in native tissue. As such, the permeability of the implantis low, thus affecting corneal hydration and overall nutritional levels.Further, since the collagen source employed can be derived from nonhumansources, there is a susceptibility to immunologic effects.

European Patent No. 339,080/EP A1 to Gibson, Lerner, et al., reveals animproved prosthetic corneal implant in that the surface of the polymeris coated with crosslinked or uncrosslinked fibronectin. While thiscoating does improve epithelial adhesion, the problems of lack ofdiffusibility, optical clarity, and foreign body rejection are stillpresent.

It is known to inject specialized gels in an effort to improve or changethe radius of curvature of the cornea. U.S. Pat. No. 5,681,869 toVillain, et al., describes a biocompatable polyethylene oxide gel forinjection into the cornea as a method of tissue augmentation. Thisprocedure suffers from the fact that any gel lacks inherent structuralintegrity and thus can only augment existing tissue through limitedhydrodynamic forces. Optical transmissibility and permeability arelimited relative to material produced by the disclosed invention.Foreign body rejection is also possible.

Several prior art references disclose means of corneal repair throughapplication of a suitable topographical ointment or solution. EuropeanPatent No. 778,021/EP A1 and Japanese Patent No. 8,133,968 JP to Ohuchiand Kato, disclose a solution of eye drops comprised of water, sodiumchloride, potassium chloride, sodium bicarbonate, and taurine. Thisproduct suffers from the fact that as essentially a simple bufferedisotonic saline solution, it is incapable of rendering any of thestructural changes in the cornea required to correct high astigmatism,keratoconus, ectasia, burns, or corneal thinning. Further, the solutionof Ohuchi and Kato is capable only of yielding temporary corneal surfacerelief due to minor, transient optical modifications.

European Patent Publication Nos. WO 00218441 and WO 00240242 to Bowlin &Wnek et al., published Mar. 7^(th), 2002 and April 8^(th) respectively,describe electrospun collagen fibers used a tissue scaffolds. Further,claims are made that the geometry of the electroprocessed matrix can becontrolled by microprocessor regulation or by moving the spray nozzlewith respect to the target or vice versa. In reality, the electriccharge that builds up on an electrospun fiber is significant, andresults in whipping effect, which can vary fiber diameter and makeprecise deposition impossible as the fiber splays about the target. Thisis because the DC high voltage source used in Bowlin et al., allows alike charge to accumulate on the fiber. As the fiber is ejected, aradius in the fiber will result in like charge repulsive forces todeflect the fiber in the opposite direction, where the radius decreasesand the repulsive force increases. This process repeats itself, leadingan uncontrolled ability to deposit material at a precise target andpattern. Further, the splaying about of the fibers results in tensileforces which varies the fiber diameter considerably.

The principal goal of the cited invention is to fabricate collagenconstructs which serve as cell growth scaffold and to encourageneovascularization or blood vessel in growth. However, cell and vesselin growth are detrimental to a successful corneal collagen fibrilstructure and if allowed to transpire, would result in blindness.Finally, the precise fibril diameter and mean spacing between suchfibrils in that construct necessary for corneal use is not described inBowlin et al. And the lack of such exact fibril specification, uniformdiameter, and matrix pattern would result in reduced opticaltransparency of the material and insufficient permeability for ocularuse.

OBJECTS AND ADVANTAGES

The disclosed invention overcomes many of the limitations inherent incorneal transplants, solid polymer implants, mechanical implantsemployed to distort or reinforce the cornea, and much more, includingthe following:

(a) It provides a means of producing collagen polymer scaffolds inorganized fibril strands at the same diameter as natural corneal stromalcollagen, assuring the same optical clarity and diffusioncharacteristics as the original tissue. Significantly, this processpermits additional tissue to be added to the cornea to augmentstructural integrity, therein correcting astigmatism, ectasia, failedLCAP, keratoconus, and other corneal problems.

(b) It affords a means of arranging collagen fibrils into a specificgeometric matrix, which accurately mimics natural corneal stromalcollagen.

(c) It teaches a means to affix the specialized collagen polymer matrixto the surrounding stromal tissue using glycerose, thereby precludingcorneal cap displacement and enhancing the structural integrity of thestroma.

(d) It reduces or eliminates corneal nerve damage as a consequence ofmicrokeratome corneal cap creation during LCAP or other similar cornealsurgical procedures through the use of polyethylene glycol.

(e) It yields a means of producing a viable collagen polymer refractivecorrecting lens whose characteristics duplicate natural tissue and iscapable of being integrated into and compatible with, the surroundingcorneal collagen. This tissue is refractive and is ablatable for LCAPoptimization.

(f) It teaches a means to create corneal collagen fibrils of thediameter, spacing, and pattern that mimics native tissue, necessary forproper transparency and hydration of the comea.

Further objects and advantages will become apparent from a considerationof the ensuing description and accompanying drawings. REFERENCE NUMERALS10 Polymer Jet 20 Taylor Cone 30 Needle 40 Hydrostatic Pump 50Collector-Target 60 Splaying Polymer Fibrils 70 Power Supply 80 PositiveHigh Voltage 90 High Voltage Return-Ground 100 Table Displacement 110Polymer Deposition Pattern

DETAILED DESCRIPTION—FIGS. 1 to 7

FIG. 1 illustrates in detail human corneal stromal collagen fibrilsobtained by scanning electron microscopy. A preferred electrosprayoperation is illustrated in FIG. 2. An electrospray needle 30. Theneedle 30 supports a Taylor Cone 20 as a result of the electric fieldbetween the source needle 30 and an oppositely charged target orelectrode. If the needle 30 were connected to a positive terminal 80 ofa suitable high voltage supply 70, then the target 50 would be thenegative terminal 90. The resulting polymer jet 10 is produced at theapex of the Taylor Cone, and the jet 10 is attracted to and acceleratestoward, the target 50 electrode. The solvent evaporates during theflight from the source needle 30 to the target 50, leaving behind asolid collagen fiber. The distance between the source needle 30 and thetarget 50 may be reduced significantly if the electrospinning isperformed in a bath of co-current or counter current gas flow, whichserves to increase the evaporation of the solvent species. The sameimprovement in evaporation may be achieved if the electrospinningapparatus is placed in a suitable chamber under partial pressure. FIG. 3illustrates a typical polymer pattern produced with a shortsource-to-target distance and a high polymer concentration. FIG. 4illustrates fine collagen fibrils by increasing source to targetdistance as opposed to that cited in FIG. 3. FIG. 5 identifies theconditions which affect electrospinning fibril diameter and fiberdensity.

A preferred embodiment of the collagen electrospinning process isillustrated in FIG. 6. A metering pump 40 exerts hydrostatic pressure ona collagen solution. Alternating current at a high voltage preferablybetween 1,000 and 20,000 volts at terminal 80, creates an electrostaticfield between the source needle 30 and the target screen 50. At the tipof the syringe needle, a Taylor cone 20 is formed which emits a finepolymer jet 10 that expands outward to produce a filament 60 anddeposited on target 50. FIG. 7 depicts the creation of an electrospunorthogonal matrix, achieved by fixing either needle 30 with respect to amoving target 50, or vice versa. When emitting jet 10, the source ortarget is displaced linearly until maximum coverage is achieved, atwhich point the direction is reversed and the process repeated,effectively “scanning” over target 100. This process is duplicated inthe alternate axis by rotating the target orthogonally. A regular matrix110 can thus be created. A final polymer “mat” is cut by several means,preferably by laser trimming.

It is imperative that in order to form collagen fibrils of the correctdiameter, preferably 65 um, and the correct mean distance, preferably300 um, that the charge on the fiber due to the electric field beneutralized. If DC is used as a high potential source, the distributedcharge will result in repulsive forces which bend the fiber being spun.This bending or whipping action repeats since like charges repel, andarc in the fiber brings these like charges closer together forcing thefiber violently in the opposite direction. The violent motion of acharged fiber exherts significant tensile loads on the fiber itself,pulling the fiber into a smaller diameter. (Use of a DC power supply maybe acceptable if the charge on the fiber is removed by other means, suchas through the use of a high voltage field emission electron source, anAC or DC corona discharge, ultraviolet light source, radioactive iongenerator).

Therefore, accurate and repeatable fibril diameters cannot be achieved.While this does not present it self as a problem for tissue scaffolding,uncontrolled fibril diameter, deposition density, and interfibrilspacing are critical parameters if a successful corneal tissue is to beachieved. Fibers that are too large in diameter will diffract incidentlight, making the material less transparent. If the fibers are too closetogether or the density of the fibril mat too great, the diffusionproperties of the resulting electrospun mat will be impaired, possiblyresulting in blindness.

The disclosed invention provides a means to augment existing cornealtissue, adding refractive material which can be subjected to LCAPoptimization. It further enhances the structural integrity of weakcornea by providing an additional collagen matrix to existing tissue andby the use of glycerose, which cross links the collagen polymer toexisting tissue. The disclosed invention is not a tissue scaffold in thetraditional sense. That is, the collagen fibrils produced by thedisclosed invention do not encourage cellular in growth orneovascularization, both of which would defeat the purpose of use in theeye by occulding light and reducing permeability of the cornealstructure.

A particular object of the invention is to provide a means of restoringto normal comeas' whose surface has been damaged by trauma, failed LCAP,burns, and other mechanical disruptions, so that optical distortion,and/or reduction of transparency is reduced or eliminated. Diseases thatimpact the cornea include keratoconus, keratoglobus, pellucid marginaldegeneration, and corneal dystrophies. The potential to either augment(as in keratoconus) or replace (as in corneal dystrophies such as Fuch'sEndothelial Dystrophy) corneal tissue is the object of this invention.

Still other objectives and possible applications of the invention willbecome evident to those knowledgeable in the related arts. The first ofwhich is the ability to create a natural corneal refractive lens to beimplanted into existing stromal tissue.

The following example illustrates the practice of the invention in apreferred embodiment. The disclosed procedure offers a means ofreconstructing corneal tissue, rebuilding stromal integrity, and cornealreshaping by laser surgery. According to the present invention, aprocess known as “electrospinning” is used to produce human collagen,preferably Type I (Type I is the principal component of bone, skin, andtendon), in micro strands that approximate or match the nanometer sizefibrils of natural human corneal stromal collagen. The fibril diameteris regarded as the principal factor in achieving corneal transparency,as does the mean distance between fibrils. The fibril strands aredeposited onto an appropriate target, which allows a collagen fibril matto develop. The density and configuration of this mat determine thepermeability of the structure to aqueous fluids, lipids, and gases, aswell as the ultimate optical transparency. The density and orientationof these fibrils, illustrated in the drawings, are controlled in orderto achieve the desired diffusive and optical parameters compatible withnatural tissue. The resulting sheet or pad of collagen fibers can betrimmed to the desired dimensions and can either be inserted under acorneal cap during normal LCAP surgery to prevent ectasia (a distensionof the cornea due to thinning), or can be placed as an corneal overlayto add structural reinforcement to the cornea in treating such disordersas keratoconus. Or, it can be used either intracorneally or topically asa refractive correcting contact lens which can absorbed and integratedinto the native corneal stromal tissue.

The Corneal Stroma

The principal structural material of the cornea is collagen; asindicated, its particular organization accounts for the transparency ofthe stroma. In the human cornea, collagen fibers have a uniform diameterand regular spacing between them. The fibers and the keratocytes betweenthem are oriented in parallel to form lamellae (or layers). The lamellaeare superposed with others in a regular order, the collagen in eachlamella being perpendicular to the adjacent lamellae. An importantfactor in transparency is the hydration of the proteoglycans(non-collagenous component of a cartilage matrix). This determines theregular spacing of the collagen fibers and the distance between thefibers. The principal keratan sulfate proteoglycans are lumican,keratocan, and mimecan.

The galactosaminoglycans rich proteoglycans (chondroitin sulphate,dermatan sulphate, and keratan sulfate) that are expressed in the stromahave a high water affinity. Their water affinity is counterbalanced bythe pump mechanisms in the endothelial cells. Proteoglycans also play arole binding the growth factors, and act as adhesive proteins. Thedifferentiated connective tissue in the stroma contains 80% to 90% ofwater on a weight basis. Collagen, other proteins, andglycosaminoglycans of mucopolysaccharides constitute the major part ofthe remaining solids. Corneal fibrils are neatly organized and presentthe typical 64 to 66 nanometer periodicity of collagen. These collagenfibrils form the skeleton of the corneal stroma. The physicochemicalproperties of corneal collagen do not differ from those of tendon andskin collagen. Like collagen from these other sources, corneal collagenis rich in nitrogen, glycine, proline, and hydroxyproline.Mucopolysaccharides (MPS; glycosaminoglycans) represent 4% to 4.5% ofthe dry weight of the cornea. MPS are localized in the interfibrillar orinterstitial space, probably attached to the collagen fibrils or tosoluble proteins of the cornea. The MPS in the interstitial space play arole in corneal hydration through interactions with the electrolytes andwater. Three major MPS fractions are found in the corneal stroma:keratin sulfate (50%), chondroitin (25%), and chondroitin sulfate (25%).The interstitial fibril structure must allow the MPS to flow freely, inconcert with water and oxygen. All of this is necessary to promotecorneal health, mechanical integrity, and optical clarity.

Creating Replacement Corneal Stromal Collagen Fibrils

Transparent stromal structures that can be implanted into a recipientcornea to augment or replace existing tissue are fabricated according tothe present invention. It further permits creation of specializedcollagen that integrates itself with the existing surrounding tissue toform a single fully functional stroma. Additional benefits include invitro creation of complete.

In order to provide a suitable stromal structure, fibrils of collagen,again, preferably Type I, must be created and layered to form the matwhich exhibits the transparency and diffusion characteristics of healthytissue. In the preferred embodiment, an electrospinning process producesthe collagen fibrils. In this technique, the polymer underconsideration, in this case the collagen solute, is dissolved by asuitable solvent and injected under hydrostatic pressure into aconductive needle or capillary. An AC or DC potential of preferably4,000 to 12,000 volts is maintained between injection needle and asuitable target located away from the needle at a distance sufficient topreclude production of a corona or arc. The voltage is adjustedaccording the distance and desired fiber diameter and structure. Thevoltage difference between the injection needle and the target suited tothe given solvent conductivity, polymer, and flow rate, the resultingelectrostatic field at the needle tip results in the formation of whatis known as a Taylor Cone. (G. I. Taylor first described how apolarizable liquid under the influence of an electric field would form ameniscus which is a cone. Proced. Royal Society, vol. 313, pp. 453,1969.) When the field is increased, a fluid jet is emitted from the tip.Evaporation of solvent from this jet results in a polymer strand ofcollagen. This strand is attracted to, and impacts with, the groundcathode target. The accumulation of such strands creates a the mat ofcollagen fibers having a diameter ranging from tens of microns or moredown to tens of nanometers or less, depending upon the concentration andnature of solute, the conductivity and viscosity of liquid, and thepotential difference between the needle and target. It has been shown byWnek et al. of Virginia Commonwealth University (VCU) inBiomacromolecules, 2002, Vol. 3, pp. 232-238, that electrospun collagenfibers can be produced down to 100+/−40 nano meters in diameter. Calfskin dissolved in a suitable solvent was electrospun, and upontransmission electron microscopy (TEM) examination, revealed the samebanded appearance characteristic of native polymerized collagen. Variouspolymers studied yielded fiber diameters in the range of 0.1 to 10 um.Extrusion, where a polymer such as collagen is drawn through an orifice,rather than electrospinning, is an alternative in certain instances.Evaporation of solvent from this jet results in a polymer strand ofcollagen. A co-current or counter current gas flow, preferably withnitrogen, can improve the solvent evaporation so that the distancebetween the spray needle and target and the applied voltage may bereduced, permitting more accurate fiber deposition control.

Collagen mats produced by this process can have diameters up to tens ofmillimeters and thicknesses of up to hundreds of microns, depending upondeposition time. Similarly, collagen for creating a suitable corneal canbe derived from a variety of sources. In the preferred embodiment,synthetic collagen such as that manufactured by FibroGen of SanFrancisco, Calif., is dissolved by a solvent such as 1,1,1,3,3,3hexaflouro-2-propanol (HFIPA) and electrospun into a fibril diameter ofpreferably 65 nanometers, +/−50 nanometers with a mean distance betweenfibers of preferably 300 um, and layered into a mat that can be trimmedto desired final dimensions. Again, extrusion rather thanelectrospinning of the polymer is an alternative in certain instances).Laser cutting is often employed since fibril terminations must besevered and should not be excessively frayed or tangled. Tangling orfraying can affect bonding to native collagen and can vary opticaltransparency. While the resulting collagen mat consists of disorganizedfibrils, this does not interfere with required transparency or diffusioncharacteristics. The general theory for corneal transparency has to dowith the diameter of the collagen fibers in reference to the wavelengthof the incident light. Organization of the fibers appears to be of lessimportance, however, the mean distance between fibrils must becontrolled. This conclusion is supported by the fact that shark corneaexhibits regions of disorganized but roughly equally spaced fibers withrandom interfibrillar distances, yet exhibit a high degree of opticaltransparency. Use of alternating current microspun collagen fibersallows precise control of the fibril diameter. The deposition ratedetermines the interfibrillar spacing.

An alternative source of suitable corneal collagen is the autologoustransplantation of patient collagen derived from biopsy from a region orregions elsewhere in the body. A useful source can be derived frompluripotent stem cells from bone marrow. The marrow contains severalcell populations, including mesenchymal stem cells that are capable ofdifferentiating into adipogenic, osteogenic, chondrogenic, and myogeniccells. Bone marrow procurement has obvious limitations, such as extremediscomfort for the patient during harvesting, thus an alternative sourceis desirable. One source found by Zuk et al., includes autologous stemcells from human adipose tissue obtained by suction-assisted lipectomyor liposuction. Grown in vitro, a fibroblast-like population of cells ora processed lipoaspirate, which differentiate into adipogenic cells thatproduce collagen. Such cultured cells are then dissolved as previouslydescribed and electrospun or extruded for corneal use.

Electrospinning Controlled Corneal Collagen Fibril Matrices

Modification of the electrospinning process to yield a cross hatchpattern is achieved by maintaining either the needle anode fixed andmoving the cathode target, or vice versa. Under normal conditions, theelectrospun collagen fiber is splayed about by the interplay ofmechanical, hydrodynamic, and electrical forces so as to cause thepolymer strands to accumulate on the target in a random pattern. Whileordinarily this is not a problem in stromal scaffold mat construction,since the fiber diameter is the principal factor in cornealtransparency, there are instances where a regular matrix of stromalcollagen is desired. By rapidly moving the needle in a linear directionfor a fixed distance and then reversing such motion with respect to thetarget, while at the same time indexing the target utilizing a steppermotor drive or piezo stack or other such precision positioner, a seriesof relatively straight and parallel fibers may be laid down on thetarget. After the desired pattern has been achieved in one axis, thetarget may be rotated ninety degrees and the process repeated.

The outer fringes of a collagen mat matrix so created will be lessorganized than the central axis as the outer edge is where targetposition reversal occurs. However, this area can be trimmed away anddiscarded with a suitable laser. The resulting central scaffold areaexhibits a collagen structure, pattern, and diameter that closely mimicsnatural stromal collagen. If even greater accuracy is required in fibrilspacing, the distance the fiber is deposited across a moving target canbe increased and the jet shut down at the point of maximum travel. Thetarget is then indexed to the next position, the electrospinning jetreestablished, and the target rapidly moved to the opposite extreme,where the process is repeated until the maximum linear coverage area offibers in one axis of orientation is achieved. The target may then berotated ninety degrees as before and the spinning procedure repeated.

Improvements to the electrospinning process include utilizing a sourceof free ions generated electrically or from a suitable radioactivesource, to neutralize the charge on the surface of the polymer jet tominimize Coulomb repulsion and thus the extent of splaying. In addition,alternating the jet high voltage polarity at high frequency can decreaseor eliminate fiber charging so that precise fiber deposition may beachieved.

Inserting the Replacement Collagen Tissue

After the fabricated microspun or extruded collagen fibril scaffold isproduced, it is preferably laser trimmed into the desired diameter andthickness required for a given recipient. The recipient is preferablytreated with pharmaceuticals used to treat glaucoma which reduce theintraocular pressure prior to the operative procedure. Employingepithelial debridement, epithelial placement to the side (such as in theLCAP procedure), or creation of a corneal flap on the patient's targetglobe, the newly grown corneal cellular sheet is placed over the denudedcorneal stroma. Orientation of an organized parallel fibril cornealsheet and the existing natural fibril structure, if required, may beaccomplished by utilizing a polarized light and rotating the appliedcollagen sheet until a similar interference pattern is achieved.Glycerose is then applied to initiate collagen crosslinking between thecorneal tissue and the corneal sheet, thereby providing an adhesive.

If a flap has been created during LCAP, additional glycerose is addedbefore the flap is dropped, covering the repair. Further, the use ofglycerose assists in maintaining corneal flap position during healing.Since adding collagen tissue may affect corneal flap suction when such aflap is replaced because overall corneal thickness will increase,glycerose-initiated crosslinking will secure the flap and added tissuein place, preventing a lost corneal cap. Further glycerose treatmentalso minimizes or eliminates the possibility of corneal wrinkles orstriae. An added benefit is that glycerose use actually increases themechanical integrity of the cornea. Experiments with rabbit eyes haveshown that corneal transparency is lost when intraocular pressure isincreased, but such is not the case with corneas similarly tested thathave been previously treated with glycerose. This fact alone holds greatpromise in effecting interstitial bonding that we believe can keepkeratoectasia (thinning of the cornea leading to distension and reducedvision) from occurring. The use of glycerose also minimizes epithelialingrowth.

Finally, the combination of glycerose with polyethylene glycol or theuse of polyethylene glycol alone can also be employed to assist in nerverepair when a corneal flap has been created. There is evidence tosupport the view that nerves severed in healthy corneas dull sensationsnecessary to effect the so called blink response, thereby potentiallycausing dry-eye syndrome. The resulting decrease in lubrication candamage the epithelial layer, increase the potential for ocularinfection, and reduce visual acuity. Polyethylene glycol (PEG) has beenshown to permit healing of recently severed nerves elsewhere in thebody, particularly for spinal nerves, but has not been reported in theliterature utilized for prevention of dry eye syndrome or any otherocular use.

After about three days, epithelial cells cover the repair site. Thedrugs employed to reduce the intraocular pressure are now discontinuedand the healing tissue is allowed to stabilize over a period of three tosix months. Corneal topographical data, wavefront measures of higherorder aberations, and other refractive measurements are then obtainedand laser reshaping subsequently performed to effect final refractivecorrection.

1. A method of producing microstrands of collagen, comprising: forming asolution of collagen using a solvent, spraying said solution of collagenonto a conductive target from a conductive spraying needle that ismaintained at a high potential with respect to said target moving saidneedle to and fro with respect to said target, allowing said solvent toevaporate from said target so as to form collagen fibrils or strands onsaid target upon the evaporation of said solvent, and continuing saidspraying until a mat of said fibrils or strands are formed on saidtarget.
 2. The method of claim 1, further including implanting said matof said fibrils into the cornea of an animal.
 3. The method of claim 2wherein said animal is human.
 4. The method of claim 1 where the sprayedfiber is exposed to ionizing radiation.
 5. The method of claim 1 wherethe electrical potential is alternated.
 6. The method of claim 2 whereglycerose is applied.
 7. The method of claim 2 where polyethylene glycolis applied.
 8. A method of claim 1 where the distributed charge of thefiber is neutralized by a source of free ions.
 9. A method of claim 8where the ion source is electrically generated.
 10. A method of claim 8where the ion source is radioactively generated.
 11. A method of claim 8where the ionizing source is an ultraviolet light.